View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Minireview 223 Firefly luciferase: the structure is known, but the mystery remains Thomas O Baldwin The structure of firefly luciferase reveals a new protein its starting condition. Firefly luciferase, on the other hand, fold which may be representative of a growing family of catalyzes an oxidative reaction involving ATP, firefly homologous enzymes. luciferin and molecular oxygen, yielding an electronically excited oxyluciferin species [2]. This excited species Address: Department of Biochemistry and Biophysics and emits visible light, which is employed by the firefly in its Department of Chemistry, Center for Macromolecular Design, reproductive behavior [3]. Firefly luciferase was one of the Texas A&M University, College Station, TX 77843–2128, USA. first enzymes to be investigated in biochemical detail [4]. Structure 15 March 1996, 4:223–228 WD McElroy and his colleagues [5–9], as well as other investigators [10] working during the 1940s and 1950s © Current Biology Ltd ISSN 0969-2126 with P. pyralis, determined the structures of the substrates and products of the enzyme. Evidence for the chemical From the Atlantic Ocean to the eastern face of the Rocky mechanism proposed for the firefly luciferase reaction [11] Mountains, a summer sunset is usually accompanied by has been discussed in a recent review [12]. one of Nature’s most delightful light shows. Photinus pyralis, the North American firefly, has entertained count- The structures of luciferases less observers, probably since the coming of humans to The structure of luciferase from the firefly P. pyralis has the continent. During the period between the 1950s and now been determined at high resolution and is reported by the 1980s, numerous young biologists earned their spend- Conti et al. in this issue of Structure [13]. As beautifully ing money as firefly collectors, first employed by Professor described in their paper, the enzyme folds into two distinct William D McElroy, then of Johns Hopkins University, domains, a large N-terminal domain comprising residues and later as members of the Sigma Firefly Club. In 1985, 4–436, and a C-terminal domain formed from residues when Marlene DeLuca and her colleagues cloned the 440–544. The structure is shown in Figure 1. The fold cDNA encoding the luciferase of P. pyralis, an alternative assumed by the luciferase polypeptide appears to be source of the enzyme became available, and soon after, in unique. The N-terminal domain consists of a b barrel and laboratories around the world, numerous other organisms two b sheets flanked by a helices which form a five-layered began to emit the characteristic yellow-green lumines- ababa structure. The C-terminal domain, consisting of cence as a consequence of expression of the firefly five b strands and three a helices, is folded into a compact luciferase in their cells. structure that is connected to the N-terminal domain by a disordered loop (connecting residues 435 and 441). There Historical perspective are three other disordered loops not visible in the electron Luciferase is a generic term describing any enzyme that density, one in the C-terminal domain connecting residues catalyzes a reaction yielding visible light. Light emission is 523 and 529, and two in the N-terminal domain (connect- a consequence of formation of a product or intermediate in ing residues 198–204 and residues 355–359). an electronically excited state; return to the ground state occurs via emission of a photon of light. Luciferases are The structure presented is without bound substrates or highly diverse, catalyzing a great variety of reactions and other ligands. Conti et al. have taken advantage of the using widely different substrates [1]. All have in common homology of firefly luciferase with other enzymes that cat- the involvement of oxygen. Luciferases are more different alyze similar reactions [14] to deduce the location of the than, for example, proteases, which all carry out hydrolytic active center (see Fig. 2). It is assumed that regions of chemistry on peptide bonds. Luciferases all emit light, but greatest sequence conservation are most likely to be they do so by very different means. Therefore, the involved in the catalytic mechanism of the enzyme. Based luciferases from different organisms probably evolved on their extensive analysis, it is proposed that the active independently, rather than from a common ancestral center is composed of residues on the surfaces of both enzyme. Bacterial luciferase, the first luciferase to be domains, and that upon substrate binding, the domains cloned and also the first to be structurally characterized, is move together to form the active center. The drawings pre- a flavin monooxygenase that utilizes flavin mononu- sented and the comparisons made with the amino acid cleotide (FMN) to activate molecular oxygen, yielding a sequences of the large family of homologous proteins con- flavin C4a peroxide. Reaction of the peroxide with an stitute a compelling argument. For a bioluminescence reac- aliphatic aldehyde substrate yields, ultimately, the car- tion to occur with a high quantum yield, as is the case for boxylic acid and the flavin C4a hydroxide in the first firefly luciferase, it is essential that water be excluded from singlet excited state. Light emission, loss of the C4a the active site. It would appear that the two-domain struc- hydroxide and dissociation of FMN returns the enzyme to ture presented by Conti et al. could serve such a purpose. 224 Structure 1996, Vol 4 No 3 Figure 1 Two orthogonal stereoviews of the surface of firefly luciferase, depicting an ‘anvil and hammer’ motif. Both views show the large N-terminal ‘anvil’ domain below the smaller C-terminal ‘hammer’ domain. The two views are the front view (top) and the right side view (bottom), obtained by rotating the front view through 90° to the left about the vertical axis. The color coding is the same as that used by Conti et al. [13], and indicates the domains and subdomains. The C-terminal domain (yellow) is the only domain composed exclusively of residues that are contiguous in the amino acid sequence, residues 440–544. The three subdomains of the N-terminal anvil consist of stretches that are not contiguous within the overall sequence. Subdomain A (blue) consists of residues 77–222 and 399–405. Subdomain B (purple) consists of residues 22–70 and 236–351. Subdomain C (green) consists of residues 4–10, 363–393 and 418–434. It appears that the active center comprises residues between the anvil and hammer, and it is suggested that the active center forms by movement of these two domains together following substrate binding [13]. (Figure courtesy of Peter Brick.) The structure of bacterial luciferase has also been deter- appears reasonable to propose that both luciferases exclude mined to high resolution without bound ligands [15]. Bac- water from their respective excited emitters by a confor- terial luciferase is a heterodimer composed of homologous mational rearrangement that occurs upon or following subunits, a and b (see [16] for a review of the bacterial substrate binding. luciferase literature). Both subunits assume the well- known (b/a)8, or TIM barrel structure, and are packed Reaction catalyzed by firefly luciferase together by a parallel four-helix bundle. The active center Firefly luciferase catalyzes a multistep reaction [21]. In is confined to the a subunit, and it has been proposed to the first step, luciferin (compound I; Fig. 3) reacts with reside within a large internal cavity which opens through a Mg2+-ATP to form luciferyl adenylate (compound II) and narrow crevice [17]. The opening to the cavity lies pyrophosphate. The luciferyl adenylate is oxidized by beneath a long disordered loop which appears to undergo molecular oxygen, with the intermediate formation of the a conformational rearrangement upon flavin binding and cyclic peroxide, a dioxetanone (compound III), and a mol- catalysis [16–20]. It has been proposed that this conforma- ecule of AMP. The dioxetanone is decarboxylated as a tional change may be necessary to exclude water from the result of intramolecular conversions (compound IV) to intermediates and the excited state of the flavin. Thus, it produce an electronically excited state of oxyluciferin in Minireview Luciferases Baldwin 225 Figure 2 Alignment of the amino acid sequences, reported by Devine et al. [49], luciferase from P. pyralis. The extent of conservation at each position is of the luciferases of Photinus pyralis (P.p), Luciola mingrelica (L.m), indicated by the following color code: red=fully conserved in all six L. cruciata (L.c), L. lateralis (L.l), and the green-emitting strain of the sequences; pink=2 different amino acids; green=3 different amino click beetle (CbG). Also shown in this alignment is the acids, and blue=4–6 different amino acids. At positions where a 4-coumarate:CoA ligase (CoA). References to the sequences are deletion has occurred in one or more of the proteins, the given in [49]. The numbering at the top refers to the sequence of the corresponding residues in the other proteins are shown in black. the enol or keto form (compound V). Return to the ground cruciata and Luciola lateralis [24], have been purified and state is accompanied by emission of a quantum of visible characterized. The L. mingrelica and L. cruciata luciferases light with a wavelength of maximum light intensity (Imax) are similar to the P. pyralis luciferase, with Imax of of 562–570 nm. Shimomura et al. [22] demonstrated that 562–570 nm, whereas the reaction catalyzed by L. lateralis one oxygen atom of the product CO2 arises from the sub- luciferase emits green light (Imax 552 nm). All Luciola strate oxygen. Non-enzymatic oxidation of luciferin yields luciferases inactivate rapidly at low ionic strength [21], oxyluciferin without luminescence [23].